JP7417950B2 - Passage time calculation device, passage time calculation method, and program - Google Patents

Description

The present invention relates to a passage time calculation device , a passage time calculation method, and a program.
This application claims priority based on Japanese Patent Application No. 2018-193875 filed in Japan on October 12, 2018, the contents of which are incorporated herein.

Regenerative medicine requires a large amount of pluripotent stem cells. The process of increasing the number of target cells is called the expansion culture process, but in the expansion culture of pluripotent stem cells such as induced pluripotent stem cells (iPS cells), the multipotency of pluripotent stem cells, that is, the undifferentiated state. It is necessary to expand the culture while maintaining the For this reason, cells that form colonies such as iPS cells are expanded and cultured by passage of the colonies. Pluripotent stem cells, which are iPS cells, change from an immature state to a mature state suitable for subculture during the culture process, and the state of the colony changes during this process. In the expansion culture step, the timing of subculture is determined based on changes in the state of the colony (referred to as "degree of colony maturity" in this application). Conventionally, the degree of maturity has been determined by an operator visually observing the size of cells within the colony, but this may vary depending on the operator.

In order to suppress variations between operators regarding the timing of subculturing, cells are collected after expansion culture to make a cell suspension, the number of cells per unit amount of cell suspension is measured, and the measurement results are An apparatus has been proposed that automatically performs the work of diluting cells to a desired cell concentration based on the cell concentration and passaging the cells (Patent Document 1). However, the device described in Patent Document 1 detaches the cells from the culture container and collects them in a collection bag, then measures and adjusts the cell number and performs subculturing, so it is difficult to determine whether the colony has matured sufficiently. It is not possible to determine whether the culture is sufficient, and if it is insufficient, the culture cannot be continued to mature the colony. There is a need for a method that can determine the maturity of adherently cultured colonies and predict the timing of subculture.

International Publication No. 2016/013394

In order to solve the above problem, one aspect of the present invention is to image the pluripotent stem cells over time in a pluripotent stem cell culture step, and to determine the colony of the pluripotent stem cells from a plurality of acquired images. a colony region extraction unit that extracts a region; and a region where a streaky pattern appears from the image of the colony region and which has a high contrast between the gaps between the plurality of pluripotent stem cells and extracts the region as an extraction target region. a muscle region extraction section; and a temporal change calculation section that calculates a temporal change in the area occupied by the extraction target region with respect to the colony region from a plurality of microscopic images taken of the pluripotent stem cells at different times ; a change point detection unit that detects a change point in a temporal change in the area occupied by the extraction target region with respect to the colony region ; and a passage time calculation unit that determines a passage time of the pluripotent stem cells based on the change point. It is a subculture period calculation device comprising:
One aspect of the present invention includes an image acquisition unit that images the pluripotent stem cells over time in a pluripotent stem cell culture step and acquires a plurality of images, and a colony area of the pluripotent stem cells that is determined from the images. Colony region extraction unit to extract, and streak region extraction to extract a high contrast region between the gaps of the plurality of pluripotent stem cells, which is a region where a streak pattern appears from the image of the colony region, as an extraction target region. a temporal change calculation unit that calculates a temporal change in the area occupied by the extraction target region with respect to the colony region from a plurality of images in which the pluripotent stem cells are captured at different times ; a change point detection unit that detects a change point in the area occupied by the extraction target region over time; a passage time calculation unit that calculates a passage time of the pluripotent stem cells based on the change point; A subculture period calculation device includes an output unit that outputs a subculture period.

In order to solve the above problem, one aspect of the present invention is to image the pluripotent stem cells over time in a pluripotent stem cell culture step, and to determine the colony of the pluripotent stem cells from a plurality of acquired images. a colony region extraction step of extracting a region; and a region where a streaky pattern appears from the image of the colony region and which has a high contrast between the gaps between the plurality of pluripotent stem cells is extracted as an extraction target region. a muscle region extraction step, and a time change calculation step of calculating a time change in the area occupied by the extraction target region with respect to the colony region from a plurality of microscopic images taken at different times of the pluripotent stem cells; A change point detection step of detecting a change point in the time change of the area occupied by the extraction target region with respect to the colony region , and a passage time calculation step of determining a passage time of the pluripotent stem cells based on the change point. A subculture period calculation method includes the following steps .

In order to solve the above problem, one aspect of the present invention allows a computer to image the pluripotent stem cells over time in a pluripotent stem cell culture step, and to determine the pluripotent stem cells from a plurality of acquired images. a colony region extraction step of extracting a colony region of stem cells; and a region to be extracted, which is a region where a streak pattern appears from the image of the colony region and has a high contrast between the gaps between the plurality of pluripotent stem cells. and a time change calculation step of calculating a time change in the area occupied by the extraction target region with respect to the colony region from a plurality of microscopic images taken of the pluripotent stem cells at different times. a change point detection step of detecting a change point of a time change in the area occupied by the extraction target region with respect to the colony area ; and a step of detecting a change point of a time change in the area occupied by the extraction target region with respect to the colony region, and a step of determining a passage time of the pluripotent stem cells based on the change point. This is a program for executing the surrogate period calculation step.

FIG. 1 is a diagram showing an example of the configuration of a subculture period calculation device according to a first embodiment of the present invention. It is a figure showing an example of a phase contrast image concerning a 1st embodiment of the present invention. It is a figure showing an example of processing of the subculture time calculation device concerning a 1st embodiment of the present invention. FIG. 3 is a diagram showing an example of a temporal change in the ratio of the area occupied by a muscle region to a colony region (muscle region ratio) according to the first embodiment of the present invention. It is a figure showing an example of subculture time calculation processing concerning a 1st embodiment of the present invention. It is a figure showing an example of composition of a subculture time calculation device concerning a 2nd embodiment of the present invention. It is a figure showing an example of subculture time calculation processing concerning a 2nd embodiment of the present invention. It is a figure showing an example of composition of a subculture time calculation device concerning a 3rd embodiment of the present invention. It is a figure showing the 1st example of subculture time calculation processing concerning a 3rd embodiment of the present invention. It is a figure which shows the 2nd example of the subculture time calculation process based on the 3rd Embodiment of this invention. FIG. 3 is a diagram showing an example of a striped pattern according to the first embodiment of the present invention.

(First embodiment)
Embodiments of the present invention will be described in detail below with reference to the drawings. FIG. 1 is a diagram showing an example of the configuration of a subculture period calculation device 1 according to the present embodiment. The passage period calculation device 1 extracts an extraction target region, which is a region to be extracted, from a plurality of phase contrast images PS (not shown) in which pluripotent stem cells are imaged. Here, pluripotent stem cells refer to stem cells that have the potential to differentiate into all three cell lineages (endoderm, mesoderm, and ectoderm) (pluripotency) and also have proliferative ability. Examples include iPS cells and embryonic stem cells (ES cells).

The plurality of phase contrast images PS are a set of time-lapse images obtained by photographing colonies of pluripotent stem cells at predetermined time intervals, and one of the phase contrast images PS is referred to as a phase contrast image P0. An example of the phase difference image P0 is shown in FIG. 2.
In this embodiment, the extraction target area is an area in the phase difference image P0 in which a streaky pattern appears. Here, in order to explain the streaky pattern, the culture process of pluripotent stem cells, which are iPS cells, will be explained.
During the culture process, pluripotent stem cells, which are iPS cells, have an initial state of non-colony formation, an immature state, and then a mature state.

The non-colony-forming state is a state in which the pluripotent stem cell is a single cell and does not form a colony. In an immature colony state, a colony is formed, but the area per cell is large and the cell density is low. Hereinafter, the region with low cell density will also be referred to as a coarse region.

The mature state is a state in which pluripotent stem cells form colonies and mature. As pluripotent stem cells mature, the area per cell in the colony decreases and the cell density increases. Hereinafter, a region with high cell density will also be referred to as a dense region. In a mature colony, a dense region is formed in the center, and a coarse region exists surrounding the dense region.

There is a transition state as an intermediate state between the colony immature state and the mature state. The transition state is a state in which pluripotent stem cells form colonies but are not fully mature.

In the transition state, a muscle region is seen in the phase contrast image P0 of the colony. The streak area is an area where a plurality of elongated streak patterns can be seen in the phase contrast image P0. It is thought that as the pluripotent stem cells mature, the pluripotent stem cells become denser with each other, and as a result, the contrast between the gaps between the multiple pluripotent stem cells becomes high, giving them a streak-like appearance. FIG. 11 shows an example of the striped pattern SS.
In the transition state, the colony includes a muscle region, a dense region surrounding the muscle region, and a coarse region surrounding the dense region.

Here, with reference to FIG. 2, the muscle region SR will be explained.
FIG. 2 is a diagram showing an example of a phase contrast image P0 obtained by imaging an iPS cell (healthy human peripheral blood-derived iPS cell line 003 created at Kyoto University iPS Cell Research Institute) according to the present embodiment. . In FIG. 2, muscle regions SR1, muscle regions SR2, and muscle regions SR3 are shown as examples of muscle regions SR included in the colony region CR of the phase contrast image P0.
The colony region CR is a region excluding the abnormal region AR among the regions corresponding to colonies of pluripotent stem cells in the phase contrast image P0.

The abnormal region AR is a region formed by abnormal cells that deviate from the state of pluripotent stem cells among the regions corresponding to colonies of pluripotent stem cells. When culturing pluripotent stem cells, it is important to maintain their pluripotent state, but abnormal cells that deviate from the pluripotent stem cell state may appear during the culture process. be.

Returning to FIG. 1, the description of the subculture time calculation device 1 will be continued.
The passage time calculation device 1 includes a phase contrast image acquisition section 10, a colony region extraction section 11, a muscle region extraction section 12, a time change calculation section 13, a change point detection section 14, and a reduction rate calculation section 15. , a passage period calculation section 16, an output section 17, and a storage section 18.

The phase difference image acquisition unit 10 acquires a plurality of phase difference images PS supplied from the phase difference image supply unit 2. Here, the plurality of phase contrast images PS are an example of a plurality of microscopic images of pluripotent stem cells captured by transmitted illumination at different times. The plurality of phase contrast images PS are time-lapse images in which the pluripotent stem cells are imaged at different times by converting the phase difference of the transmitted light of the illumination light irradiated onto the pluripotent stem cells into a brightness difference. . In the plurality of phase contrast images PS, for example, enlarged images of pluripotent stem cells obtained by a phase contrast microscope are captured.
The multiple phase contrast images PS are multiple images of pluripotent stem cells taken at regular time intervals.
The microscopic image obtained by transmitted illumination may be, for example, a differential interference image, a quantitative phase contrast image, etc., in addition to a phase contrast image.

The colony region extraction unit 11 extracts a colony region CR of pluripotent stem cells from each phase contrast image P0.

The muscle region extraction unit 12 extracts the muscle region SR from the colony region CR in each phase difference image P0. The streak region SR is an example of an extraction target region, and is a region where a streak pattern appears in the phase contrast image P0.
Here, the streak region extracting unit 12 can extract the streak region SR based on the streak-like high-intensity region, for example. The high brightness area is an area based on a collection of pixels having a brightness value larger than a reference value among the pixels forming the phase difference image P0. The high-luminance area includes a collection of pixels with a luminance value greater than a reference value and the surrounding area of the collection of pixels.

The temporal change calculation unit 13 calculates a temporal change G1 in the area occupied by the muscle region SR for the plurality of phase difference images PS. In this embodiment, the temporal change calculation unit 13 first calculates the muscle area ratio A in each phase difference image P0. Here, the muscle region ratio A is the ratio of the area occupied by the muscle region SR to the colony region CR.
The time change calculation unit 13 calculates the time change G1 of the muscle area ratio A in the plurality of phase difference images PS based on the time when each phase difference image P0 was captured and the muscle area ratio A at that time. It is stored in the storage unit 18 as .

The change point detection unit 14 detects the change point M1 of the time change G1 calculated by the time change calculation unit 13. Here, the change point M1 is a point indicating the time when the ratio of the area of the muscle region SR to the area of the entire colony region CR increases once and then begins to decrease. The change point M1 is, for example, a point where the slope of the time change G1 changes from positive to negative. That is, the change point M1 is the maximum value of the time change G1.

The present inventors found that when a colony of pluripotent stem cells is cultured from an immature state to a mature state, the ratio of the area of the muscle region SR to the area of the entire colony region CR (muscle region ratio A) once increases. It was found that the muscle area SR decreased and eventually almost disappeared. Therefore, it is possible to judge the maturity level based on the change in the muscle area ratio A.

The reduction rate calculation unit 15 calculates, with respect to the time change G1 calculated by the time change calculation unit 13, a reduction rate D of the area occupied by the muscle region SR with respect to the colony region CR after the change point M1 detected by the change point detection unit 14. Calculate.

In this embodiment, the reduction rate calculation unit 15 calculates the reduction rate D of the muscle area ratio A after the change point M1 with respect to the time change G1. Here, the reduction rate D is, for example, the amount of reduction in the muscle area ratio A per unit of time.

The passage time calculation unit 16 calculates the passage time PT of the pluripotent stem cells based on the change point M1.
In this embodiment, the passage time calculation unit 16 calculates the passage time PT of the pluripotent stem cells based on the reduction rate D and the predetermined threshold TH. In this embodiment, the passage time calculation unit 16 calculates the time when the muscle area ratio A becomes equal to or less than the threshold value TH indicated by the threshold value information 181 from the reduction rate D as the passage time PT. Here, the threshold information 181 is information indicating a predetermined threshold of the muscle area ratio A.

The output unit 17 outputs the passage time PT to the presentation unit 3 and causes the presentation unit 3 to display the passage time PT. Note that the output unit 17 may output the passage time PT to an output device other than the presentation unit 3, a storage device, or the like.
The storage unit 18 stores time change information 180 and threshold information 181.

The phase difference image supply unit 2 supplies a plurality of phase difference images P0 to the subculture period calculation device 1. The phase contrast image supply unit 2 is, for example, an imaging device equipped with a phase contrast microscope.
The presentation unit 3 presents the passage time PT supplied from the passage time calculation device 1. The presentation unit 3 is, for example, a display device such as a display.

Next, with reference to FIGS. 3 to 5, the process by which the passage time calculation device 1 calculates the passage time PT will be described.
FIG. 3 is a diagram showing an example of processing of the subculture period calculation device 1 according to the present embodiment.
Step S100: The subculture time calculation device 1 starts the process of calculating the subculture time PT for each imaging time of a plurality of phase difference images PS. Here, in this embodiment, the number N of frames of the plurality of phase difference images PS is determined in advance. The passage time calculation device 1 repeats the process of calculating the passage time PT a predetermined number of frames N.

Step S110: The phase difference image acquisition unit 10 acquires the phase difference image P0 supplied from the phase difference image supply unit 2. Here, the phase difference image supply unit 2 supplies the captured phase difference image P0 to the subculture period calculation device 1 every time the phase difference image P0 is captured.
The phase difference image acquisition section 10 supplies the acquired phase difference image P0 to the colony region extraction section 11 and the muscle region extraction section 12.

Step S120: The colony region extraction unit 11 extracts the colony region CR of pluripotent stem cells from the phase contrast image P0 acquired by the phase contrast image acquisition unit 10. The colony region extraction unit 11 extracts a cell region from the phase contrast image P0 based on known edge detection, for example, and extracts a cell region larger than a predetermined area from the extracted cell region as a colony region CR. The colony region extraction section 11 supplies the extracted colony region CR to the muscle region extraction section 12 and the time change calculation section 13.
Note that extraction of the colony region CR is not limited to the method based on the area of the cell region, and can be performed by various known methods.

Here, the colony region extracting unit 11 may extract, as the colony region CR, a region excluding the abnormal region AR based on the presence or absence of a surrounding halo.
A halo is a part of a phase difference image in which the phase difference between one area and the other area is large, and as a result, the brightness at the boundary between one area and the other area is higher than the surrounding area. A colony of normal pluripotent stem cells has many halos around it, and a colony of abnormal cells has few halos around it.

Therefore, the colony region CR can be a region in which a colony is formed that has a portion around it where the phase difference is greater than or equal to a predetermined value. The abnormal region AR can be a region of the colony region CR that does not have a surrounding portion where the phase difference is greater than or equal to a predetermined value.

Step S130: The muscle region extraction unit 12 extracts the muscle region SR from the colony region CR in the phase contrast image P0. Here, the streak region extraction unit 12 can extract the streak region SR based on the streak-like high-intensity region. The muscle region extraction section 12 supplies the extracted muscle region SR to the time change calculation section 13.

Step S140: The time change calculation unit 13 calculates a time change G1 in the area ratio (muscle region ratio A) occupied by the muscle region SR to the colony region CR.

The temporal change calculation unit 13 first calculates the muscle area ratio A for each phase difference image P0. Here, for convenience, among the plurality of phase difference images PS, the phase difference image P0 acquired this time is referred to as the phase difference image _Pn , and the phase difference images acquired so far are referred to as the phase difference images _P0 to Pn _-1. do. The time change calculation unit 13 acquires the time change information 180 calculated based on the phase difference images P ₀ to P _n−1 from the storage unit 18, and applies the time change G1 indicated by the acquired time change information 180 to the phase difference image By adding a set of the imaging time of P _n and the calculated area ratio, a temporal change G1 based on the phase difference images P ₀ to P _n is calculated.
The temporal change calculation unit 13 stores the temporal change G1 calculated based on the phase difference images P ₀ to P _n in the storage unit 18 as the temporal change information 180. Further, the temporal change calculation unit 13 supplies the calculated temporal change G1 to the change point detection unit 14.

As described above, the temporal change calculation unit 13 calculates the temporal change G1 in the muscle area ratio A from the plurality of phase difference images PS.

Step S150: The change point detection unit 14 detects the change point M1 of the temporal change G1.

Now, with reference to FIG. 4, the time change G1 will be explained.
FIG. 4 is a diagram showing an example of a temporal change G1 in the ratio of the area occupied by the muscle region SR to the colony region CR (muscle region ratio A) according to the present embodiment. In the time change G1, the muscle area ratio A is shown for each time when the phase difference image P0 is captured.
Note that the example of the time change G1 shown in FIG. 4 is virtual data simulating the time change G1 of the muscle area ratio A.

In the time change G1 shown in FIG. 4, the muscle area ratio A increases up to point X1 and begins to decrease from point X1. The change point detection unit 14 compares the muscle area ratio A at adjacent points and determines that the muscle area ratio A increases up to point X1, and decreases for the first time from point X1 to point X2. In this case, the point X1 is detected as the change point M1.

Further, the change point detection unit 14 may detect the change point M1 of the time change G1 as a point where the slope of the time change G1 changes from positive to negative. In this way, in this embodiment, the change point M1 can be detected easily.

In addition, when the muscle area ratio A is decreasing at three or more consecutive points of the time change G1, the change point detection unit 14 detects one of the three or more consecutive points (for example, the most (new point) may be detected as the change point M1. Here, the plurality of points being continuous in the time change G1 means that there is no imaging time corresponding to a point other than the plurality of points during the imaging time corresponding to the plurality of points. In other words, the times when the plurality of phase difference images PS are captured are continuous as frames.

For example, the change point detection unit 14 detects that for consecutive points X1, X2, and X3 in the time change G1, the muscle area ratio A at point X2 decreases from the muscle area ratio A at point X1, and the muscle area ratio A at point X3 If the area ratio A is decreasing from the muscle area ratio A at the point X2, the point X1 may be detected as the change point M1.
In the passage period calculation device 1, it is easier to determine whether the muscle area ratio A has decreased based on three consecutive points than to determine whether the muscle area ratio A is decreasing based on two consecutive points. The accuracy of M1 detection can be increased.

Further, when the slope of the time change G1 is smaller than a predetermined negative value, the change point detection unit 14 detects a point corresponding to the earliest imaging time among the points in the time change G1 used to calculate the slope. may be detected as the change point M1.

Note that two or more points in the temporal change G1 that the change point detection unit 14 uses to detect the change point M1 do not need to be consecutive.

Returning to FIG. 3, the explanation of the processing of the subculture period calculation device 1 will be continued.
Step S155: The change point detection unit 14 determines whether or not the change point M1 has been detected. If the change point M1 is detected in step S150, the change point detection unit 14 determines that the change point M1 has been detected, and if the change point M1 is not detected in step S150, it is determined that the change point M1 has not been detected. judge.

When determining that the change point M1 has been detected (step S155; YES), the change point detection unit 14 supplies change point information indicating the detected change point M1 to the reduction rate calculation unit 15. After that, the subculture period calculation device 1 executes the process of step S160.
On the other hand, when the change point detection unit 14 determines that the change point M1 has not been detected (step S155; NO), the subculture time calculation device 1 executes the process of step S180.

Step S160: The passage time calculation unit 16 and the reduction rate calculation unit 15 perform a passage time calculation process.
Here, with reference to FIG. 5, the subculture time calculation process will be described.
FIG. 5 is a diagram illustrating an example of the subculture time calculation process according to the present embodiment.

Step S200: The reduction rate calculation unit 15 calculates the reduction rate D of the muscle area ratio A after the change point M1 with respect to the time change G1. Here, "after the changing point M1" refers to the range of imaging time after the imaging time of the phase difference image P0 corresponding to the changing point M1.
The reduction rate calculation unit 15 supplies the calculated reduction rate D to the subculture period calculation unit 16.

Here, with reference to FIG. 4 again, the process by which the reduction rate calculation unit 15 calculates the reduction rate will be described.
As an example, the reduction rate calculation unit 15 calculates the difference between the muscle area proportion A corresponding to the point X2 and the muscle area proportion A corresponding to the point X1 to the imaging time of the phase difference image P0 corresponding to the point X2 and the point X1. The reduction rate of the muscle area ratio A is calculated by dividing by the difference from the imaging time of the corresponding phase difference image P0. That is, in this embodiment, the reduction rate D is the value of the slope of the straight line connecting the point X1 and the point X2.

Note that the reduction rate calculation unit 15 may calculate the reduction rate D of the muscle area ratio A based on a plurality of points after the change point M1 in the temporal change G1. The plurality of points after the change point M1 are one or more points after the point X2 in addition to the point X1 and the point X2.

Returning to FIG. 5, the explanation of the subculture period calculation process will be continued.
Step S210: The passage time calculation unit 16 determines the passage time PT of the pluripotent stem cells based on the reduction rate D calculated by the reduction rate calculation unit 15 and the threshold value TH indicated by the threshold information 181 acquired from the storage unit 18. Calculate.

Here, if the passage time PT calculated in the process of step S210 from the second time onward is different from the passage time PT calculated in the previous process of step S210, the passage time calculation unit 16 calculates the newly calculated passage time PT. The passage time PT is corrected by the time PT. The passage time calculation unit 16 calculates the corrected passage time PT as the passage time PT.
The passage time calculation unit 16 supplies the calculated passage time PT to the output unit 17.

In the passage time calculation device 1, the process of calculating the passage time PT is repeated and continued by the number of frames N of the plurality of phase difference images PS, so the passage time PT once calculated can be corrected. The accuracy can be increased.

Here, with reference to FIG. 4 again, the process by which the passage time calculation unit 16 calculates the passage time PT will be described.
The passage time calculation unit 16 calculates the time when the muscle area ratio A becomes equal to or less than the threshold value TH based on the reduction rate D. Here, the threshold value TH is 15 percent, for example. The passage time calculation unit 16 sets the calculated time as the passage time PT.

As described above, the reduction rate D is the value of the slope of the straight line connecting the point X1 and the point X2. The passage time PT is calculated based on approximation by a linear equation of the time change G1.

Note that the subculture time calculation unit 16 may calculate an approximate curve that approximates the range after the change point M1 of the time change G1 using a plurality of points after the change point M1 in the time change G1. The passage time calculation unit 16 may calculate the imaging time of the phase difference image P0 at which the muscle area ratio A becomes equal to or less than the threshold TH based on the calculated approximate curve.

Returning to FIG. 3, the explanation of the processing of the subculture period calculation device 1 will be continued.
Step S170: The output unit 17 outputs the passage time PT calculated by the passage time calculation unit 16 to the presentation unit 3, and causes the presentation unit 3 to display the passage time PT.
Step S180: When the subculture time calculation device 1 has executed the process of calculating the subculture time PT for each imaging time of the plurality of phase difference images PS for the number of frames N, it ends the process.

In the present embodiment, a case has been described in which the phase difference image supply unit 2 supplies the captured phase difference image P0 to the subculture period calculation device 1 every time the phase difference image P0 is captured. Not exclusively. The phase difference image supply unit 2 may supply a plurality of captured phase difference images P0 to the subculture time calculation device 1 at a predetermined timing.

As described above, the subculture time calculation device 1 according to the present embodiment includes the time change calculation section 13, the change point detection section 14, and the subculture time calculation section 16.
The time change calculation unit 13 calculates a region in which a streaky pattern appears from a plurality of microscopic images (in this example, a plurality of phase contrast images PS) in which pluripotent stem cells are imaged by transmitted illumination at different times. A time change G1 in the area (muscle area ratio A in this example) occupied by a certain extraction target region (in this example, muscle area SR) is calculated.

With this configuration, the subculture time calculation device 1 according to the present embodiment can predict the timing of subculture, so that it is possible to subculture at an appropriate timing. The present inventors have found that when a colony of pluripotent stem cells passes a mature state and reaches a supermature state, engraftment and proliferation after subculture are poor. However, according to the passage timing calculation device 1 according to the present embodiment, by setting an appropriate threshold value TH, it is possible to predict the optimal passage timing, and the pluripotent stem cells in culture are not overly mature. can reduce the risk of

Furthermore, the subculture period calculation device 1 according to the present embodiment includes a reduction rate calculation section 15.
The reduction rate calculation unit 15 calculates the reduction rate D of the area after the change point M1 (muscle area ratio A in this example) with respect to the time change G1.
The passage time calculation unit 16 calculates the passage time PT based on the reduction rate D and a predetermined threshold TH.

With this configuration, the passage time calculation device 1 according to the present embodiment can calculate the reduction rate D of the muscle area ratio A and calculate the passage time PT based on the calculated reduction rate D. The accuracy of calculating the passage time PT can be increased compared to the case where it is not calculated.

(Second embodiment)
A second embodiment of the present invention will be described in detail below with reference to the drawings.
In the first embodiment, the passage time calculation device calculates the passage time based on the reduction rate of the muscle area ratio A and a predetermined threshold value. In this embodiment, a case will be explained in which the passage time calculation device calculates the passage time based on passage time information in which change points and passage times are associated in advance for each type of pluripotent stem cell. do.
The passage time calculation device according to this embodiment is referred to as a passage time calculation device 1a.

FIG. 6 is a diagram showing an example of the configuration of the subculture time calculation device 1a according to the present embodiment. Comparing the passage time calculation device 1a (FIG. 6) according to the present embodiment and the passage time calculation device 1 (FIG. 2) according to the first embodiment, it is found that the passage time calculation unit 16a and the storage unit 18a are different. Here, other components (phase contrast image acquisition section 10, colony region extraction section 11, muscle region extraction section 12, time change calculation section 13, change point detection section 14, reduction rate calculation section 15, and output section 17) The functions it has are the same as those in the first embodiment. Description of the same functions as in the first embodiment will be omitted, and in the second embodiment, the description will focus on parts that are different from the first embodiment.

The passage time calculation unit 16a calculates the passage time PT based on the change point M1 and the passage time information 181a. The passage time information 181a is information in which the change point M1 and the passage time PT are associated in advance for each type of pluripotent stem cell.

The storage unit 18 stores time change information 180 and passage time information 181a.
Here, the passage time information 181a includes, for example, the change point M1 and the change point M1 for each type of pluripotent stem cell based on the process (FIG. 3) in which the passage time calculation device 1 described in the first embodiment calculates the passage time. The passage time PT is calculated in advance and stored in the storage unit 18.

Next, with reference to FIG. 7, the process by which the passage time calculation device 1a calculates the passage time PT will be described. In the process in which the subculture time calculation device 1a calculates the subculture time PT and the process in which the subculture time calculation device 1 of the first embodiment calculates the subculture time (FIG. 3), the subculture time calculation in step S160 Processing is different.

FIG. 7 is a diagram illustrating an example of the subculture time calculation process according to the present embodiment.
Step S300: The passage time calculation unit 16a acquires the passage time information 181a from the storage unit 18a.

Step S310: The passage time calculation unit 16a calculates the passage time PT based on the change point M1 and the passage time information 181a. Here, the passage time calculation unit 16a selects information corresponding to the type of pluripotent stem cells from the passage time information 181a, and reads out the passage time PT corresponding to the change point M1 from the selected information. Calculate the passage time PT.

As explained above, in the passage time calculation device 1a according to the present embodiment, the passage time calculation unit 16a calculates the change point M1 detected by the change point detection unit 14 and the change point M1 detected by the change point detection unit 14, and The passage time PT is calculated based on the passage time information 181a in which the change point M1 and the passage time PT are associated in advance.

With this configuration, the passage time calculation device 1a according to the present embodiment performs passage based on the passage time information 181a in which the change point M1 and the passage time PT are associated in advance for each type of pluripotent stem cell. Since the time PT can be calculated, the processing load can be reduced compared to the case where it is not based on the subculture time information 181a.

In addition, in this embodiment, the passage time information 181a is such that the change point M1 and the passage time PT are associated in advance for each type of pluripotent stem cell, but the passage time information 181a is The change point M1 and the passage time PT may be associated in advance with the type and number of competent stem cells.

(Third embodiment)
Hereinafter, a third embodiment of the present invention will be described in detail with reference to the drawings.
In the first embodiment and the second embodiment described above, a case has been described in which the passage time calculation device repeats and continues the process of calculating the passage time by the number of frames of a plurality of phase difference images. In this embodiment, a case will be described in which the subculture time calculation device cancels the process after calculating the subculture time. Furthermore, in this embodiment, a case will be described in which the passage time calculation device changes the time interval at which pluripotent stem cells are imaged.
The passage time calculation device according to this embodiment is referred to as a passage time calculation device 1b.

FIG. 8 is a diagram showing an example of the configuration of the subculture time calculation device 1b according to the present embodiment. Comparing the passage time calculation device 1b (FIG. 8) according to the present embodiment and the passage time calculation device 1 (FIG. 2) according to the first embodiment, it is found that the imaging end instruction section 19b and the imaging time interval change section 20b is different. Here, other components (phase contrast image acquisition section 10, colony region extraction section 11, muscle region extraction section 12, time change calculation section 13, change point detection section 14, reduction rate calculation section 15, subculture time calculation section 16 and the output unit 17) have the same functions as in the first embodiment. Description of the same functions as in the first embodiment will be omitted, and in the third embodiment, the description will focus on parts that are different from the first embodiment.

When the passage time PT is calculated by the passage time calculation unit 16, the imaging end instruction unit 19b supplies the phase contrast image supply unit 2 with an end instruction indicating the end of imaging of the pluripotent stem cells.
After the change point M1 is detected by the change point detection unit 14, the imaging time interval change unit 20b sends an imaging time change instruction to the phase contrast image supply unit 2 to increase the time interval at which pluripotent stem cells are imaged. supply to.

Next, with reference to FIG. 9, the process by which the passage time calculation device 1b calculates the passage time PT will be described. The process in which the subculture time calculation device 1b calculates the subculture time PT and the process in which the subculture time calculation device 1 of the first embodiment calculates the subculture time (FIG. 3) include the subculture time calculation process in step S160. are different.
Note that in the process in which the subculture time calculation device 1b calculates the subculture time PT, the number of frames of the plurality of phase difference images PS does not need to be determined in advance.

With reference to FIG. 9, the subculture time calculation process of the subculture time calculation device 1b will be described.
FIG. 9 is a diagram showing a first example of the subculture period calculation process according to the present embodiment. Note that each process of step S400 and step S410 is the same as each process of step S200 and step S210 in FIG. 5, and therefore a description thereof will be omitted.
Step S420: The imaging end instruction section 19b supplies an end instruction to the phase difference image supply section 2. That is, the imaging end instruction unit 19b instructs the end of imaging of pluripotent stem cells when the passage time PT is calculated by the passage time calculation unit 16.

The phase difference image supply section 2 ends the time-lapse imaging based on the end instruction supplied from the imaging end instruction section 19b.
In the passage time calculation device 1b, imaging of pluripotent stem cells can be completed when the passage time PT is calculated, so that the data amount of the plurality of phase contrast images PS can be reduced.

Next, with reference to FIG. 10, another example of the passage time calculation process of the passage time calculation apparatus 1b will be described.
FIG. 10 is a diagram showing a second example of the subculture time calculation process according to the present embodiment. Note that each process of step S510 and step S520 is the same as each process of step S200 and step S210 in FIG. 5, and therefore a description thereof will be omitted.
Note that, before starting the passage time calculation process in FIG. 10, the changing point detection unit 14 supplies the imaging time interval changing unit 20b with information indicating that the changing point was detected in step S155 in FIG. 2.

Step S500: The imaging time interval changing unit 20b supplies an imaging time changing instruction to the phase difference image supplying unit 2. That is, the imaging time interval changing unit 20b changes the time interval at which the pluripotent stem cells are imaged after the changing point M1 is detected by the changing point detecting unit 14.
The phase difference image supply unit 2 lengthens the imaging time interval for time-lapse photography from the length before the change, based on the imaging time change instruction supplied from the imaging time interval changing unit 20b.

Note that the imaging time interval changing unit 20b may supply the imaging time changing instruction to the phase difference image supplying unit 2 after the passage time PT is calculated by the passage time calculating unit 16. That is, the imaging time interval changing unit 20b may change the time interval at which the pluripotent stem cells are imaged after the passage time PT is calculated by the passage time calculation unit 16.

Note that, when the muscle area ratio A calculated by the time change calculation unit 13 is greater than or equal to a predetermined ratio, the imaging time interval changing unit 20b changes the imaging time interval for time-lapse photography so that the muscle area ratio A is greater than or equal to a predetermined value. It may be shortened depending on the length of the imaging time interval before . The predetermined ratio here is, for example, zero. That is, when the muscle region SR appears in the plurality of phase difference images PS, the imaging time interval changing unit 20b shortens the length of the imaging time interval after the muscle region SR appears.

In order to calculate the passage time PT, the time change G1 after the appearance of the muscle region SR is used, so the passage time calculation device 1b calculates the length of the interval between imaging times after the appearance of the muscle region SR. By shortening , the accuracy of calculating the passage time PT for the number of frames N of the plurality of phase difference images PS can be increased.

Furthermore, the imaging time interval changing unit 20b makes the imaging time interval of the time-lapse photography before the muscle area ratio A calculated by the time change calculating unit 13 exceeds a predetermined ratio longer than the predetermined length of time. It's fine.

In order to calculate the passage time PT, the time change G1 after the appearance of the muscle region SR is used, so the passage time calculation device 1b calculates the time change G1 of the imaging time before the appearance of the muscle region SR. By making the interval longer than the predetermined length of time, the amount of data of the plurality of phase difference images PS before the muscle region SR appears can be reduced.

As described above, the subculture time calculation device 1b according to this embodiment includes the imaging time interval changing section 20b. The imaging time interval changing unit 20b changes the time interval at which the pluripotent stem cells are imaged after the changing point M1 is detected by the changing point detecting unit 14.

With this configuration, the passage time calculation device 1b according to the present embodiment can lengthen the time interval at which pluripotent stem cells are imaged after the change point M1 is detected, so that the amount of data of the plurality of phase contrast images PS can be increased. It can be reduced.

Additionally, the present inventors have also found that colonies in which a muscle region appears have a high probability of maturing thereafter, and colonies in which a muscle region is not observed have a high probability of not maturing. Therefore, the subculture period calculation devices 1, 1a, and 1b in the embodiments described above can be used in cases where the muscle region extraction unit 12 does not extract a muscle region within a predetermined period, or when the area of the muscle region does not reach a predetermined area. In this case, the display unit 3 may have a function of determining that the culture is to be discontinued and displaying the determination result on the presentation unit 3.

Note that some of the passage time calculation devices 1, 1a, and 1b in the embodiments described above, such as the phase contrast image acquisition section 10, the colony region extraction section 11, the muscle region extraction section 12, the temporal change calculation section 13, and the change inspection The output unit 14, the reduction rate calculation unit 15, the subculture time calculation units 16, 16a, the output unit 17, the imaging end instruction unit 19b, and the imaging time interval change unit 20b may be realized by a computer. In that case, a program for realizing this control function may be recorded on a computer-readable recording medium, and the program recorded on the recording medium may be read into a computer system and executed. Note that the "computer system" herein refers to a computer system built into the passage period calculation apparatus 1, 1a, 1b, and includes hardware such as an OS and peripheral devices. Furthermore, the term "computer-readable recording medium" refers to portable media such as flexible disks, magneto-optical disks, ROMs, and CD-ROMs, and storage devices such as hard disks built into computer systems. Furthermore, a "computer-readable recording medium" refers to a medium that dynamically stores a program for a short period of time, such as a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line. In this case, it may also include a device that retains a program for a certain period of time, such as a volatile memory inside a computer system that is a server or a client. Further, the program may be one for realizing a part of the above-mentioned functions, or may be one that can realize the above-mentioned functions in combination with a program already recorded in the computer system.
Further, a part or all of the subculture period calculation devices 1, 1a, and 1b in the embodiments described above may be realized as an integrated circuit such as an LSI (Large Scale Integration). Each of the functional blocks of the subculture period calculation devices 1, 1a, and 1b may be individually implemented as a processor, or some or all of them may be integrated into a processor. Further, the method of circuit integration is not limited to LSI, but may be implemented using a dedicated circuit or a general-purpose processor. Further, if an integrated circuit technology that replaces LSI emerges due to advances in semiconductor technology, an integrated circuit based on this technology may be used.

Although one embodiment of the present invention has been described above in detail with reference to the drawings, the specific configuration is not limited to that described above, and various design changes may be made without departing from the gist of the present invention. It is possible to

DESCRIPTION OF SYMBOLS 1, 1a, 1b... Passage time calculation device, 2... Phase difference image supply part, 3... Presentation part, 10... Phase contrast image acquisition part, 11... Colony region extraction part, 12... Muscle region extraction part, 13... Time Change calculation section, 14... Change point detection section, 15... Decrease rate calculation section, 16, 16a... Passage time calculation section, 17... Output section, 18, 18a... Storage section, 180... Time change information, 181... Threshold information , 181a... Passage time information, 19b... Imaging end instruction section, 20b... Imaging time interval changing section

Claims (11)

a colony region extraction unit that images the pluripotent stem cells over time in a pluripotent stem cell culturing step and extracts a colony region of the pluripotent stem cells from a plurality of acquired images;
a streak region extracting unit that extracts, as an extraction target region, a region where a streak pattern appears from the image of the colony region and which has a high contrast between the gaps between the plurality of pluripotent stem cells;
a temporal change calculation unit that calculates a temporal change in the area occupied by the extraction target region with respect to the colony region from a plurality of microscopic images taken of the pluripotent stem cells at different times ;
a change point detection unit that detects a change point in the area occupied by the extraction target region over time with respect to the colony region ;
a passage time calculation unit that determines the passage time of the pluripotent stem cells based on the change point;
A passage time calculation device comprising: a reduction rate calculation unit that calculates a reduction rate of the area after the change point detected by the change point detection unit with respect to the time change in the area calculated by the time change calculation unit;
Furthermore,
The passage time calculation device according to claim 1, wherein the passage time calculation unit calculates the passage time based on the reduction rate calculated by the reduction rate calculation unit and a predetermined threshold value. The streak region extracting unit extracts the extraction target region from the colony region based on a high brightness region that is a region based on a collection of pixels having a brightness value larger than a reference value among pixels forming the image,
The passage time calculation unit generates passage time information in which the change point detected by the change point detection unit is associated in advance with the change point and the passage time for each type of pluripotent stem cell. The passage time calculation device according to claim 1, wherein the passage time is determined based on the following. an image acquisition unit that images the pluripotent stem cells over time in the pluripotent stem cell culturing step and acquires the plurality of images ;
Furthermore ,
The temporal change calculation unit calculates a temporal change in the area occupied by the extraction target region, which is a region in which the striped pattern appears, by calculating an area of the extraction target region with respect to an area of the colony region in the image. The subculture period calculation device according to any one of claims 1 to 3, wherein the subculture period is calculated as a time change in a proportion occupied by the period. 5. The method according to claim 1, further comprising: an imaging time interval changing unit that changes a time interval at which the pluripotent stem cells are imaged after the change point is detected by the change point detection unit. The passage time calculation device described. The passage time calculation device according to claim 2, wherein the passage time calculation unit is capable of changing the predetermined threshold value. an image acquisition unit that images the pluripotent stem cells over time in a pluripotent stem cell culturing process and acquires a plurality of images;
a colony region extraction unit that extracts a colony region of the pluripotent stem cells from the image;
a streak region extraction unit that extracts a high contrast region between the gaps between the plurality of pluripotent stem cells, which is a region where a streak pattern appears from the image of the colony region, as an extraction target region;
a temporal change calculation unit that calculates a temporal change in the area occupied by the extraction target region with respect to the colony region from a plurality of images in which the pluripotent stem cells are captured at different times ;
a change point detection unit that detects a change point in the area occupied by the extraction target region over time with respect to the colony region ;
a passage time calculation unit that calculates the passage time of the pluripotent stem cells based on the change point; an output unit that outputs the passage time;
A passage time calculation device comprising: The passage time calculation device according to claim 7, further comprising a display unit that displays the passage time outputted from the output unit. The streak region extracting unit extracts the extraction target region from the colony region based on a high brightness region which is a region based on a collection of pixels having a brightness value larger than a reference value among pixels constituting the image. Ru
The passage time calculation device according to claim 7, comprising: a colony region extraction step of imaging the pluripotent stem cells over time in the pluripotent stem cell culturing step and extracting a colony region of the pluripotent stem cells from a plurality of acquired images;
a streak area extraction step of extracting an area where a streak pattern appears from the image of the colony area and a high contrast area between the plurality of pluripotent stem cells as an extraction target area;
a time change calculation step of calculating a time change in the area occupied by the extraction target region with respect to the colony region from a plurality of microscopic images taken at different times of the pluripotent stem cells;
a change point detection step of detecting a change point in the area occupied by the extraction target region over time with respect to the colony region ;
a passage time calculation step of determining the passage time of the pluripotent stem cells based on the change point;
A method for calculating the passage period. to the computer,
a colony region extraction step of imaging the pluripotent stem cells over time in the pluripotent stem cell culturing step and extracting a colony region of the pluripotent stem cells from a plurality of acquired images;
a streak area extraction step of extracting an area where a streak pattern appears from the image of the colony area and a high contrast area between the plurality of pluripotent stem cells as an extraction target area;
a time change calculation step of calculating a time change in the area occupied by the extraction target region with respect to the colony region from a plurality of microscopic images taken at different times of the pluripotent stem cells;
a change point detection step of detecting a change point in the area occupied by the extraction target region over time with respect to the colony region ;
a step of calculating the passage time of the pluripotent stem cells based on the change point;
A program to run.

Images